Efficient communication between motor neurons and muscle fibers depends on the establishment and maintenance of the neuromuscular junction (NMJ). NMJ assembly requires the muscle-specific receptor tyrosine kinase, MuSK, which is activated by neurally-secreted Agrin. Agrin binds to low-density lipoprotein receptor-related protein-4 (Lrp4), stimulating association between Lrp4 and MuSK and MuSK activation. In addition to promoting NMJ stability by mediating Agrin-induced MuSK activation, Lrp4 also acts directly on nerve terminals, possibly through a soluble extracellular fragment released by proteolysis, to induce presynaptic differentiation. Mechanisms of MuSK activation and Lrp4 signaling are complex and not fully understood. The proposed work will probe structural transitions that govern Agrin-Lrp4-MuSK signaling, both in vitro and in vivo, in order to advance the long-term goal of understanding the crucial molecular interactions in this pathway. This broad objective will be pursued through three specific aims: (1) Characterize Agrin-induced conformational changes in Lrp4 and MuSK that facilitate MuSK activation; (2) Characterize conformational changes in MuSK induced by binding of an agonist antibody; and (3) Determine whether cleavage of Lrp4 is required for presynaptic differentiation. These studies will utilize fluorescence-based methods and X-ray crystallography to characterize conformational changes in the extracellular domains of Lrp4 and MuSK that are induced by binding to Agrin, as well as those induced by a MuSK agonist antibody. The proposed work will also explore the mechanism and consequences of proteolytic shedding of the extracellular region of Lrp4 for presynaptic differentiation. Detailed structural insights into the actions of MuSK and Lrp4 at the NMJ should reveal how this complex regulates synaptic differentiation and suggest avenues for therapeutic intervention in neuromuscular disease.